利用生物資訊方法探討在水稻中受過氧化氫調控的小核醣核酸

Abstract

在植物體內，過氧化氫為一種強的氧化劑，其通常是受到生物性與非生物性逆境的誘導而產生。過去的研究也認為過氧化氫可以作為二次的訊息傳遞物質以調控植物的生長與逆境的防禦反應。小分子核醣核酸是一種長度約20到24個核苷酸大小的小片段非編碼核糖核酸，利用後轉錄調控機制影響植物的生理功能。然而，我們對於受到過氧化氫調控的小分子核醣核酸與其調控何種下游目標基因仍未明朗，因此本研究主要使用生物資訊方法闡明與探索過氧化氫可能調控的分子機制。本研究使用小RNA定序法與已發表之AGO1蛋白免疫共沉澱定序法所產生的序列資料分析受過氧化氫調控的小分子核醣核酸。並且使用psRNAtarget軟體與已發表的微陣列資料預測受小分子核醣核酸調控的目標基因，從中我們成功預測出377個小分子核醣核酸與其目標基因的調控路徑。為了更進一步確認小分子核醣核酸與其預測之目標基因是否存在真實的調控關係，我們開發了一個嶄新的分析軟體Pycleave對於水稻降解組進行分析，此軟體整合了psRNATarget 及 CleaveLand 。我們確認了64個mRNA在過氧化氫處理之下，可能受到小分子核醣核酸所調控。其中我們發現miR156受到過氧化氫所影響，而其目標基因為一個參與茉莉酸訊息調控的重要基因OsTIFY11b。此調控路徑我們使用即時定量聚合酶鏈鎖反應、農桿菌滲入短暫共表現法、已發表的miR156大量表現微陣列資料、轉基因植物法進行驗證。我們的結果闡明了一個嶄新的小分子核醣核酸與其目標基因的調控網絡。另一方面，我們亦對於小RNA定序法發現的轉運核糖核酸衍生之小核醣核酸進行分析。我們的結果顯示了由轉運核糖核酸衍生之小核醣核酸可能受到氧化逆境所累積。此外，轉運核糖核酸衍生之小核醣核酸可能不是RNA降解的副產物。總體而言，過氧化氫廣泛的調控小核醣核酸。其分子機制值得進一步探討。

Hydrogen peroxide (H2O2) is a strong oxidizer, whose production is commonly enhanced by abiotic and biotic stresses in plants. It has also been considered as a signal messenger involved in regulating growth and defense in plants. MicroRNAs (miRNAs) are 20-24 nucleotides non-coding RNAs playing crucial roles in biotic and abiotic responses by post-transcriptional regulation. However, the underlying mechanism for the regulation of H2O2-related miRNA and its targets remains unclear. In this study, differentially expressed miRNAs via small RNA sequencings and AGO1 immunoprecipitation-sequencings were discovered. Program psRNAtarget was used to predict miRNA target and the predicted targets were also analyzed by the published cDNA microarray data from rice treated with H2O2. Herein, 377 predicted miRNA-targets were discovered. In order to gain insight into the cleavage site of miRNA-target, a new program Pycleave was developed to analyze degradome data. The combination of psRNATarget and CleaveLand indicated that 64 mRNAs may be regulated by miRNAs. Among them, miR156 was regulated by H2O2. the predicted target gene of miR156 was OsTIFY11b, which is a member of JAZ protein, and may be involved in jasmonic acid signal transduction. This miRNA-target module was validated by real-time RT-PCR, agroinfiltration, microarray data from rice overexpressing miR156, and transgenic approaches. Our results may reveal a new miRNA and regulatory framework involved in H2O2 signal transduction. On the other hand, tRNA-derived small RNAs (tsRNAs) were also investigated in this study. Our results suggested tsRNAs were accumulated during oxidative stress. Moreover, tsRNAs might not be the byproducts from RNA degradation. Overall, H2O2 controlled small RNAs widely. The underlying molecular mechanisms for small RNAs regulated by H2O2 is worth for further study.